Molecular mechanisms of ammonium transport and accumulation in plants

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1 FEBS Letters 581 (2007) Minireview Molecular mechanisms of ammonium transport and accumulation in plants Uwe Ludewig *, Benjamin Neuhäuser, Marek Dynowski Zentrum für Molekularbiologie der Pflanzen (ZMBP), Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, Tübingen, Germany Received 31 January 2007; revised 12 March 2007; accepted 14 March 2007 Available online 22 March 2007 Edited by Ulf-Ingo Flügge and Julian Schroeder Abstract The integral membrane proteins of the ammonium transporter (AMT/Rh) family provide the major route for shuttling ammonium ðnh þ 4 =NH 3Þ across bacterial, archaeal, fungal and plant membranes. These proteins are distantly related to the Rh (rhesus) glycoproteins, which are absent in higher plants, but are present in many species, including bacteria and mammals. It appears that the large nitrogen requirement of plants resulted in unique strategies to acquire, capture and/or release ammonium. The biological function of plant ammonium transporters will be discussed and compared to other AMT/Rh proteins. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Nitrogen; Rhesus glycoprotein; Gas channel; Ammonia toxicity 1. Introduction Nitrogen is a major nutrient for all organisms, but its availability is limited in many natural ecosystems. Plants are well adapted to low nitrogen soils and frequently ammonium is the preferred nitrogen source [1]. Localized nitrogen supply stimulates root growth, root branching and lateral root elongation, but most of the positive effects on root proliferation have been attributed to nitrate, rather than ammonium. Nevertheless, plants clearly benefit from the uptake of ammonium, which typically is in the range of lm in agricultural soils, but its essentiality is contrasted by its toxicity when in excess [1,2]. 2. Biological relevance of AMT ammonium transport * Corresponding author. Fax: address: uwe.ludewig@zmbp.uni-tuebingen.de (U. Ludewig). Abbreviations: AMT, ammonium transporter; MEP, methylammonium permease; RhAG, Rhesus glycoprotein A; MeA, methylammonium; CLC, chloride channel; GS, glutamine synthetase; TIP, tonoplast intrinsic protein Ammonium (this term will be used for the sum of NH þ 4 and NH 3 ) is a central precursor of nucleic acids, proteins and other organic molecules, as well as a product of their catabolism. Because of the ph-dependent equilibrium between the uncharged NH 3 and the charged NH þ 4 form (pk a = 9.25), the ion is predominant under all physiological conditions. At nearly neutral ph, only 1% is in the NH 3 form. Classic studies revealed that synthetic lipid membranes are relatively impermeable to the cationic NH þ 4, but are relatively permeable for the uncharged NH 3. Transport of both species can be significant in biological membranes. The low capacity NH 3 diffusion depends on the lipid composition and the side activities of other transporters or channels that allow some ammonium leakage. The non AMT-mediated flux is typically enhanced at alkaline ph and is sufficient for ammonium nutrition of E. coli above 1 mm [3] and of the yeast Saccharomyces cerevisiae above 5mM [4]. The facilitated specific acquisition by AMT/Rh proteins is most important when passive diffusion becomes limiting for growth, for retrieval of NH 3 =NH þ 4 that is otherwise lost by leakage to the environment, or when ammonium transport must be regulated. The importance of regulated ammonium fluxes is readily realized by the fact that most eukaryotic and prokaryotic organisms sequenced so far have amt/rh genes in their genome. The few organisms that lack ammonium transporters appear to be either specialized to utilize specific nitrogen forms, such as urea in the case of Helicobacter pylori, or to grow in nitrogen-rich environments. Examples are the pathogenic eukaryotic parasites Plasmodium falciparum and Trypanosoma brucei which thrive in the blood of their obligate hosts. A comprehensive review on AMT function and regulation in bacteria, fungi and plants is available [5]. 3. Redundant and non-redundant functions of AMTs in plants The molecular basis of ammonium transport and its regulation by the nutritional status has been investigated in many plant species and strong correlative evidence links high-affinity ammonium influx to AMT expression. A recent detailed review summarizes the current molecular and physiological knowledge of plant ammonium transport [2]. We will focus here on the most recent findings and on the model plant Arabidopsis thaliana, which contains six sequences coding for AMTs in its genome. The cellular and sub-cellular localization of several Arabidopsis AMTs is known and their preferential tissue expression is given in Fig. 1. AtAMT2;1 is closely related to prokaryotic members like EcAmtB from Escherichia coli and AfAMT-1 from Archaeoglobus fulgidus. The Rh glycoproteins from mammals are distantly related to both plant and prokaryotic members (Fig. 1) /$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 2302 U. Ludewig et al. / FEBS Letters 581 (2007) Fig. 1. Unrooted sequence distance tree of selected AMT/Rh homologs based on a ClustalW alignment. The tree contains two prokaryotic AMTs with known three-dimensional structure from Archaeoglobus fulgidus and Escherichia coli (grey); all AMTs from the plant Arabidopsis thaliana and three characterized members from Lycopersicon esculentum (tomato, green) and the human Rh glycoproteins and non-glycosylated Rh proteins. The preferential site of expression is given next to the sequence names. The correlation of AtAMT-transcripts and proteins with ammonium influx established that transcriptional control is a major response to nutritional carbon or nitrogen availability and regulates the ammonium fluxes in plants [6 9]. The transcripts of four root expressed AtAMTs are upregulated by nitrogen deficiency, by photosynthetic products such as sugars and are diurnally regulated, albeit to a different extent [6,8,10]. The AtAMT1;1 transcript expression and ammonium influx were down-regulated after ammonium re-supply to N-starved plants and these effects negatively correlated with glutamine levels, suggesting that glutamine may be a feedback signal for inhibition of ammonium influx after re-supply [7]. The use of knockout alleles has confirmed that AMT proteins provide the major route of high-affinity ammonium influx in Arabidopsis roots. The ammonium influx in N-deficient roots was reduced by 30% by the loss of AtAMT1;1 [9,11]. A further reduction in ammonium uptake by 30% was observed by the additional loss of AtAMT1;3, which showed that both transporters act in an additive way under N-deficiency [9]. Consistent with a direct NH þ 4 uptake function of AtAMT1;1 and AtAMT1;3 from the soil, both proteins were plasma membrane localized and expressed in rhizodermal and cortical cells of primary and lateral roots, including the root hair zone [9,12]. In the more basal root zone, AtAMT1;1, but not AtAMT1;3, was mainly detected in the pericycle. AtAMT1;1, but not AtAMT1;3, was also strongly expressed in shoots. Surprisingly, not only the promoter region, but also the gene coding sequence of AtAMT1;1 appears to contain information for regulation by nitrogen [13]. When AtAMT1;1 was heterologously expressed in tobacco from a viral promoter (35S CaMV), this transcript was increased under nitrogen starvation [13]. Ectopic expression of a 35S::AtAMT1;1 construct in Arabidopsis allowed AMT1;1 transcript accumulation in shoots only under nitrogen deficiency, but led to transcript degradation in roots, indicating a nitrogen-independent and tissue specific regulation at the level of mrna degradation [13]. Such regulation was not observed for a 35S::AtAMT1;3 construct. Interestingly, the well established down-regulation of the AtAMT1;1 promoter activity by nitrogen in roots was contrasted by the reverse effect in shoots, where transcription was lower in nitrogen sufficiency [14]. AtAMT1;1 was subsequently expressed in Xenopus oocytes and analyzed using a two-electrode voltage clamp. The application of micromolar ammonium or the transport analog methylammonium (MeA) reversibly induced small, inwardlydirected currents [12,15]. The concentration that permitted half-maximal currents was between 2 and 34 lm for NH þ 4, largely consistent with earlier studies using NH þ 4 inhibition of 14 C-MeA uptake in a yeast mutant that contains no endogenous ammonium transporters (triple-mepd) [6,10,16]. These results are consistent with the fact that the application of low lm ammonium to Arabidopsis roots (and other plants) rapidly depolarized the membrane potential [16]. Transport of NH þ 4 (rather than of NH 3 ) is predicted to accumulate ammonium in the cell due to the negative membrane potential. The ammonium transport form was also tested using a yeast mutant strain that lacked all endogenous ammonium transporters and the vacuolar proton pump. This mutant was neither capable of acquiring MeA across the plasma membrane, nor to trap MeA + in an acidic vacuole. Consistent with transport of the charged species, expression of AtAMT1;1 in that yeast mutant allowed 14 C-MeA accumulation by at least 60- fold [15]. Despite the fact that ammonium influx under N-deficiency was reduced up to 70% by the loss of AtAMT1;1 and AtAMT1;3, only a little effect on growth was observed [9]. The single loss of AtAMT1;1 affected growth only under certain conditions that involved sole ammonium nutrition and the addition of sugars [11]. Thus under most conditions, the residual ammonium transport is sufficient for growth. In the root, the plasma membrane localized AtAMT1;2 is expressed in the root endodermis and weakly in the cortex. This distinct cellular localization may explain its somewhat altered transcriptional regulation compared to other root AMTs. AtAMT1;2 is involved in ammonium transfer into the vascular tissue, since apoplasmic ammonium diffusion across the endodermis is restricted by the casparian strip [17]. When heterologously expressed in oocytes, AtAMT1;2 elicited large currents that were half maximal at 140 lm ammonium [17]. Like AtAMT1;2 the plasma membrane protein AtAMT2;1 is also detected in roots and shoots [10]. The transcript was expressed in the vasculature, cortical and root tip cells and was de-repressed by nitrogen deprivation in roots, but not in shoots. The loss of AtAMT2;1 by RNAi did not affect plant growth [10]. In addition to the AMT-dependent high-affinity ammonium transport systems in plants, a lower affinity, non-saturable at least partially protein-mediated influx exists in plants, including Arabidopsis [7]. The molecular identity of this component is not well established, but NH þ 4 -permeable cation channels and aquaporin homologs may contribute to that flux. 4. Biophysical properties of AMT function The ionic currents elicited by atleast one AMT, LeAMT1;1 from tomato, have been analyzed in detail after heterologous expression in oocytes [18]. Oocytes show a variety of endogenous background currents that may even differ between oocyte batches. Consequently, the detection of ammonium-induced

3 U. Ludewig et al. / FEBS Letters 581 (2007) currents is not always a proof for NH þ 4 transport by the heterologously expressed protein. It is hence essential to test whether the elicited ionic currents differ from endogenous currents. The currents elicited by ammonium in LeAMT1;1- expressing oocytes differed from the endogenous currents: (i) only a few lm NH þ 4 elicited AMT currents, but many-fold higher concentrations were needed for the activation of endogenous currents. Furthermore, the saturation properties were distinct. (ii) LeAMT1;1 currents were identical between ph 5.0 and ph 8.0, but endogenous currents were strongly activated at more alkaline ph. (iii) Ammonium- and MeA-induced currents by LeAMT1;1 were inward at all membrane potentials, in contrast to the endogenous currents which were outward at positive potentials. Further evidence for NH þ 4 -specific currents by LeAMT1;1 came from another biophysical study [19]. Using labeled 14 C- MeA, the uptake and ionic currents were measured in parallel and compared in LeAMT1;1 and non-injected controls. In LeAMT1;1-expressing oocytes the current magnitude agreed with the hypothesis that each 14 C-MeA was transported with one positive electric charge, i.e. the experiments indicated net MeA + transport. By monitoring the intracellular ph (ph i )of voltage clamped oocytes with a fluorescent dye, it was shown that LeAMT1;1 strictly selects for net NH þ 4 transport, but excludes NH 3 transport. Acidification was only identified in voltage clamped, but not in unclamped oocytes, probably for two reasons: (i) NH þ 4 influx at normal resting potentials is small compared to the buffering capacity of the cytosol. At the cytoplasmic near neutral ph only 1% of the inflowing NH þ 4 is deprotonated to form H + and NH 3. (ii) The transported NH þ 4 depolarizes the cell, diminishing the driving force for NH þ 4. These experiments suggested that even residual net NH 3 transport is unlikely in LeAMT1;1. 5. Ammonium transporters from other species: Rh glycoproteins For comparison, we will briefly consider the molecular function of related proteins in their biological context. The distantly related rhesus associated glycoprotein (RhAG) from human erythrocytes was functionally analyzed by several different methods and the data pointed towards an electroneutral transport mechanism [20 23]. These data were interpreted either as NH þ 4 =Hþ antiport or channel-mediated NH 3 transport, which are biologically equivalent. The rationale for the proposed NH þ 4 =Hþ antiport mechanism was mainly based on the observation that the affinity of 14 C-MeA transport was only slightly changed by alterations in the external ph. This indicated that NH þ 4 rather than NH 3 binds and saturates the transporter. However, it is possible that NH þ 4 binds, is deprotonated and is finally conducted as NH 3. Such a transport mechanism has been suggested for the bacterial homolog EcAmtB and will be discussed later. Thermodynamic laws demand that transport is bi-directional in either case (NH þ 4 or NH 3 transport) and that the transport direction depends on the respective gradient. In the absence of strong ph gradients, as in many mammalian cells (Fig. 2A), a NH 3 channel may be ideal to facilitate flux in both directions, depending on the metabolic situation. In native membranes RhAG is a component of the heteromeric Rh complex and is essential for its plasma membrane localization. The complex has variable non-glycosylated RhCE Fig. 2. Biological function of AMT/Rh membrane proteins in ammonium transport. (A) NH 3 facilitation by RhAG in human erythrocytes. Strong consumption and catabolism of glutamine may preferentially lead to release of ammonium. (B) NH 3 -facilitation by bacterial EcAmtB followed by metabolic trap. (C) NH þ 4 transport via plant AMTs provides the main route for ammonium influx into the roots. Ammonia transfer across the tonoplast may be provided by TIP2 aquaporins. and RhD subunits, which expose the epitopes for the Rh antigens D or E/e and C/c on their external loops. The presence (Rh + ) or absence (Rh ) of RhD is of clinical importance in transfusion medicine, as genetic incompatibility can cause hemolytic disease. It is also important during pregnancy, where hemolytic disease of the newborn occurs when a Rh mother has become sensitized to Rh + blood through a prior pregnancy or transfusion, and developed antibodies against the RhD antigen. RhCE and RhD have not yet been shown to transport any substrate so far, which can be explained by the lack of essential, central pore-lining histidine residues (see below). Another homolog, the Rh glycoprotein RhBG, which is preferentially expressed in ovaries, skin, liver and kidney, facilitated ammonia and MeA transport, but did not elicit ionic ammonium currents in oocytes [24,25]. Since the function of AMT/Rh proteins was often analyzed by different methods, expression systems, variable ammonium concentrations and individual protocols, it was difficult to

4 2304 U. Ludewig et al. / FEBS Letters 581 (2007) derive solid conclusions about the transport mechanism in each AMT/Rh. To overcome this difficulty, LeAMT1;2 from tomato was recently compared with human HsRhCG using identical protocols. HsRhCG is abundant in the kidney, but is also detected in testis and brain. The comparison in oocytes and yeast showed that LeAMT1;2 facilitated electrogenic transport, which is coupled to the electrochemical gradient of NH þ 4, but HsRhCG transported along the chemical gradient of NH 3 without generating ionic currents [26]. When expressed in yeast, AMTs and Rh glycoproteins were both competent to restore growth on low ammonium [20,26]. However, AMTs are generally more efficient and are less dependent on the ph [4,20,26,27]. In addition, the growth of yeast is sensitive to high concentrations of MeA; this effect is more pronounced in wild type yeast than in the triple-mepd mutant. While AMTs increased the sensitivity to MeA, the Rh glycoproteins restored yeast growth on high MeA [20,26]. The alleviation of MeA toxicity by Rh glycoproteins may reflect the efflux of uncharged MeA, but the gradients for both MeA species are unknown. Thereby other interpretations, including compartmentalization into the vacuole, also appear possible. AMT-type transporters have not been identified in the genome of mammals, but such genes exist in lower animals. Members of the Rh-branch have not been identified in higher plants but are present in the genomes of algae and of many other organisms, including the ammonia accumulating and oxidizing bacteria Nitrosomonas europaea. 6. Prokaryotic AmtB homologs The three-dimensional structure of two prokaryotic AMTs has been determined at atomic resolution. AMTs are trimers of which each subunit consists of 11 alpha helical transmembrane helices arranged in a two-fold quasi-symmetry [28 30]. Within the center of each subunit, a highly hydrophobic non-polar pore is identified that is lined by two highly conserved histidines which form a hydrogen bond. The remarkable conservation of this imidazole pair arrangement and its importance for transport in EcAmtB was proven in a recent mutational study: only the first histidine could be replaced by another amino acid, aspartate, to yield a partially active MeA transporter [31]. The transport mechanism proposed from the structure involves NH þ 4 recruitment at the extracellular pore entrance. This chemical environment reduces the pk a of the incoming ammonium and finally NH 3 is conducted [28,29]. Hence the conversion of NH þ 4 to NH 3 precedes conduction. This conclusion was mainly based on the rigid AmtB structure that was almost unchanged when crystallized with or without substrate and on the hydrophobic character of the pore [28,29]. This elegant mechanistic interpretation of the structure was at variance with the long-standing view that prokaryotic high affinity transporters are NH þ 4 uniporters. Hence functional data were needed to confirm this hypothesis. Indeed, biochemical evidence in favor of NH 3 transport was provided by purified and lipid vesicle reconstituted EcAmtB [28]. By using AmtB and Glutamine Synthetase (GS) mutants, it was subsequently concluded that AmtB equilibrates NH 3 and that the accumulation of NH þ 4 and MeA+ is an experimental artifact that results from assimilation of both solutes by GS [32]. Importantly, another extraordinary feature of EcAmtB was also reported: that the saturation of transport most probably reflects the properties of GS [32]. AmtB did not saturate at lm concentration, indicating a high capacity, channel-like transport (Fig. 2B). Many previous reports on bacterial methylammonium transport had shown that transport and intracellular trapping as glutamylamides are experimentally distinguishable processes and that transport was blocked by a collapse of the membrane potential. For example, high affinity ammonium transport in Azotobacter vinelandii was suggested to be in the ionic form, but AvAmtB has not been molecularly analyzed [33]. These bacteria are soil diazotrophs that can grow in nitrogen limiting habitats and are able to fix nitrogen when grown aerobically. Taking into account the many similarities between A. vinelandii and E. coli, it remains a future challenge to identify why ammonium/mea transport depends on the membrane potential [32]. The transcript of EcAmtB is only present under nitrogen limiting conditions [5]. If AmtB equilibrates NH 3, cytosolic NH 3 cannot accumulate and efficient nitrogen acquisition requires powerful GS activity at very low cytosolic ammonium to form glutamine. Metabolic coupling and close co-operativity of GS and AmtB has indeed been observed in E. coli (Fig. 2B) [32]. The proposed transport mechanism for AmtB agrees with most observations on Rh glycoproteins, suggesting that prokaryotic AMTs and Rh glycoproteins share the same transport mechanism. Conversely, the data suggest that AMTs of prokaryotic and eukaryotic origin have thermodynamically antipodal mechanisms (transport is either coupled to the NH 3 or NH þ 4 gradient), despite belonging to the same family. This implies that ammonium acquisition and metabolism in E. coli is fundamentally different from ammonium uptake in plants (Fig. 2B and C). In order to understand how proteins select for a substrate such as NH 3, which is similar in size to H 2 O, it is instructive to have a look at the mechanism that glutamine synthetase (GS) uses to catalyze the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate. In this process, GS initially appears to recognize NH þ 4, which is then de-protonated to NH 3, which finally attacks the c-glutamyl phosphate intermediate that has formed at the active site. Similar as AmtB, the GS enzyme is competent to select at high rate with high affinity for NH 3 in aqueous solution [34]. 7. Lessons from the structure To learn more about the mechanism in plant AMTs, a LeAMT1;1 homology model was constructed based on the available structures [19]. Despite the limited significance of a model compared to a true structure, it was obvious that the major topology and most of the crucial residues are conserved. The pore lining residues in AmtB and in the homology model of LeAMT1;1 are shown in Fig. 3. Surprisingly, most residues that have been implicated in the NH þ 4 recruiting, NH 3 -channel mechanism in AmtB are identical. Thus, based on the homology structure, one might speculate that LeAMT1;1 should also conduct NH 3. However, as was discussed above, LeAMT1;1 transports net NH þ 4, but not NH 3 [18,19].

5 U. Ludewig et al. / FEBS Letters 581 (2007) Fig. 3. Comparison of the EcAmtB structure (A) and a homology model of LeAMT1;1 (B). The side chains of the pore lining residues in the structure (PDB code: 1U7G) of EcAmtB in comparison with the LeAMT1;1 homology model [19] are explicitly shown. Identical residues are shown in salmon, divergent but corresponding residues in green. The non-polar character of the pore-lining residues is entirely conserved in LeAMT1;1. Most differences are observed at the cytoplasmic pore exit. Despite the fact that the AmtB structures were thoroughly analyzed and convincingly interpreted, it must be noted that the prediction of a transport mechanism and of proton coupling can be an extremely hard task. This is evident from the analysis of CLC-type anion transporter structures and must be taken into consideration. Among CLCs, both channels and H + -coupled antiporters have been identified. Although individual CLC proteins transport by thermodynamically antipodal mechanisms, they share a common structure. The obligatory 2Cl /H + exchange in a prokaryotic, structurally resolved CLC, was lost by a single mutation within the pore, which led to channel-mediated Cl flux. However, the structure of this mutant was essentially identical to the wild type. The only difference was that the site occupancy by conducting anions correlated with the exchanger or channel-like mechanism [35]. It should be noted that the pore lining residues in LeAMT1;1 differ from EcAmtB in the more cytosolic region, although they are still non-polar (Fig. 3). In analogy to the mechanism proposed for AmtB, the model of LeAMT1;1 may hypothetically suggest that NH þ 4 is de-protonated within the pore. NH 3 is then conducted across a short distance and is re-protonated just behind the second histidine. A proton is conducted in a stoichiometric manner. It is possible that the pore histidines participate in H + transfer, but it is not necessarily the same H + resulting from de-protonation that associates with NH 3 to form NH þ 4. By such a mechanism, LeAMT1;1 transports NH 3 within a short constricted region and co-transports H + by an unknown pathway. Such a NH 3 /H + co-transport mechanism is highly attractive, since it can explain the excellent selectivity of AMTs against alkali cations [18]. The electrophysiological data are nevertheless equally well explained by a mechanism in which NH þ 4 is entirely conducted through the pore as the cation. Both mechanisms are consistent with a function of plant AMTs in initial uptake of NH þ 4 and in NH 3=NH þ 4 retrieval that counteracts the passive NH 3 loss that has been estimated to be large, both in roots and shoots [36]. The external recruitment site in most AMTs is composed of serine, tryptophan, phenylalanine and another aromatic residue, which can be either tryptophan or phenylalanine. All electrophysiologically investigated plant AMTs have identical residues at the recruitment site, but their affinity varies by at least a decade. Interestingly, the disruption of the aromatic site by insertion of a hydrophobic amino acid (Y133I) in LeAMT1;1 increased the affinity for NH þ 4 and MeA + by 10-fold and lowered the maximal transport capacity. That disruption of this site increased, rather than decreased, the affinity to NH þ 4 may suggest that other sites deeper in the pore are involved in the high affinity. If so, the recruitment site not only allows discrimination against water, but the moderate binding favors only brief and transient occupancy by NH þ 4 and allows high rate access into the pore lumen. The saturation of NH þ 4 (and MeA+ ) transport in LeAMT1;1 depends on the membrane potential, a finding that can easily be explained by NH þ 4 driven into a site that is buried deeply within the transmembrane electric field (30% electrical distance from the outside) [18]. Taking into account the structure of AmtB and the model of LeAMT1;1, this may suggest that NH þ 4 easily penetrates deeply into the constricted part of the LeAMT1;1 pore, possibly close to the positions of the conserved histidines. In the AMT-homolog MEP2 from yeast, the exchange of an absolutely conserved aspartate to glutamate (D186E) also affected saturation properties; this indicates that even residues more distant from the pore vestibule and pore center influence the K m [37]. Is there a biological role for the different saturation values of plant ammonium transporters? In principle, it appears counterproductive to limit the uptake rate to very low concentrations. However, high affinity binding often correlates with high-specificity and low transport capacity. Conversely, lower affinity may correlate with decreased specificity and high capacity, but currently there is no evidence for less specificity of any AMT. It is possible that the high affinity of AtAMT1;1 is an adaptation to low ammonium concentrations in the soil, but this also prevents excess ammonium transport. The strongly negative membrane potential of roots (typically more negative than 140 mv) can drive a more than 100-fold accumulation of NH þ 4 across the plasma membrane (Fig. 2C). 8. Ammonium transport in the symbiotic interaction Soil bacteria from the genus Rhizobium are able to induce differentiation and formation of symbiotic nodules on the roots of legumes. When free-living in soil under nitrogen limitation, the bacterium Rhizobium etli expresses an amtb gene [38]. In endosymbiotic, non-dividing bacteroids of Rhizobia,

6 2306 U. Ludewig et al. / FEBS Letters 581 (2007) ReAmtB does not participate in the release of NH 3, as the gene is switched off [38]. Interestingly, ectopic expression of the ReamtB gene affects and impairs the symbiosome differentiation and further nodule development [38]. The plant-derived peribacteroid membrane which surrounds the bacteroids is highly permeable to NH 3 and NH þ 4 [39]. In situ hybridization studies in the legume Lotus identified that the functional LjAMT2;1 transporter was expressed in nodule tissue [40]. The release of NH 3 may also be facilitated by NOD26, an aquaporin (water channel) homolog that is highly expressed in this membrane [39]. Further experimental evidence that aquaporin homologs are involved in NH 3 fluxes in plants will be discussed below. 9. Switching off ammonium transport Irrespective of the details of the AMT transport mechanism, some properties of the plant AMTs appear more consistent with a transporter-like but not with a channel-like transport. This includes the requirement for conformational changes of the aromatic phenylalanines (residues F137 and F259 in LeAMT1;1) in order to open the pore (Fig. 3). Further support for a transporter-like mechanism comes from the observation that transport within a single AMT subunit is dependent on its neighbors. Transport is inhibited by point mutations in the cytoplasmic carboxy-tail of co-expressed AMTs [17,41,42]. Homology models suggested that the relevant residues are positioned in a loop structure between two conserved short a-helices and lie on top of the neighboring subunit, explaining their involvement in subunit co-operativity [17,42]. Remarkably, a large-scale phospho-proteomic study identified that an AMT-derived peptide was phosphorylated in a neighboring threonine. The mutational exchange of this threonine by a charged amino acid that mimics phosphorylation inhibited NH þ 4 transport both in AtAMT1;1 and in AtAMT1;2 [17,42]. In addition, the mutations in one subunit also inhibited co-assembled wild type subunits within the trimer when co-expressed in yeast [42] or in oocytes [17]. This suggests that phosphorylation is involved in the gating of AMTs; the phosphorylation of a single carboxy-terminal residue appears sufficient to inactivate the entire trimer. Although the stimuli for (de-)phosphorylation are unknown, this posttranslational mechanism can explain why high affinity ammonium uptake in nitrogen-sufficient roots of Arabidopsis was (almost) absent, despite the fact that AMT proteins were present [9,12] and can explain the rapid decrease of ammonium influx after re-supply to N-deficient plant roots [7]. An elegant suppressor screen further identified mutations within AMTs that reverse the dominant negative inhibition by the C-terminus [42]. In a parallel study on EcAmtB, remarkable similarities regarding potential interactions between EcAmtB monomers and the role of the C-terminal region have been reported [43]. It is likely that this efficient shut-off mechanism is related to ammonium stress and toxicity. 10. Involvement of AMTs in ammonium toxicity Although there is much variation between individual species, an early plant response to exclusive millimolar ammonium nitrogen nutrition is reduced leaf expansion, while later syndromes include impaired root growth and chlorosis [44]. The mechanisms underlying ammonium toxicity are not fully understood, but the acidification of the external root surrounding medium, disequilibrium in the acid/base balance and energetic imbalance from active extrusion of NH þ 4 from the root may be key factors [45]. Furthermore, hormonal imbalance is involved and a strong decline of cytokinins in the xylem sap was reported [44]. The involvement of plant AMTs at high ammonium concentrations appears unlikely since amt genes are transcriptionally downregulated when nitrogen is high and AMTs are saturated much below. However, we cannot exclude the fact that AMTs contribute to or prevent ammonium toxicity. Indeed, a first hint for an involvement of AMTs in ammonium toxicity comes from yeast, an organism that is remarkably insensitive to the extremely high ammonium in standard laboratory growth media. DNA microarray analysis on chemostat cultures indicated that ammonium inhibited growth under potassium limitation [46]. The over-expression of ammonium transporters increased ammonium toxicity. Interestingly, the growth impairment was more severe with the over-expression of a relatively low affinity transporter, but was less pronounced with a very high affinity AMT [46]. Thus a high affinity (low capacity) transport may be beneficial with respect to ammonium toxicity. It is interesting to note that the growth supporting function of both transporters on limiting ammonium was very similar [4]. It may also be relevant to plant research that ammonium toxicity in yeast correlated with enhanced excretion of amino acids to alleviate the symptoms of toxicity [46]. 11. Ammonium transport by unrelated proteins There is increasing evidence that ammonium (or MeA) accumulates in the large vacuolar lumen, e.g. the accumulation of 14 C-MeA in yeast depends on the vacuolar V-ATPase [15,47]. MeA was not metabolized in yeast [47]. Taking into account the thermodynamic gradients across the tonoplast (Fig. 2C), NH 3 transport followed by acid trap might be involved in the loading and possibly in the re-mobilization of ammonium. In agreement with this, vacuolar tonoplast intrinsic proteins (TIPs) from wheat and Arabidopsis were identified that were capable of transporting ammonium and MeA [27,48]. The identified proteins belong to the TIP2 subfamily. Members of this subfamily are known to transport water and small, uncharged solutes such as urea, but in planta data for a physiological role in small solute transport is still missing. Results of the initial experiments were consistent with the interpretation that TIP2s conduct the uncharged form, NH 3 and MeA (Fig. 2C) [27]. Subsequent expression of TIP2;1 and several mammalian aquaporins in oocytes showed that these proteins did not only increase the NH 3 fluxes, but also increased the ionic currents at millimolar ammonium [49]. It has hence been discussed that these currents by ammonium might reflect NH þ 4 transport through the TIP pores themselves, in addition to NH 3 transport [49]. This topic may need further analysis in the future, keeping in mind that Rh glycoproteins, which appear to facilitate NH 3, elicited similar, strongly phdependent electrical currents in oocytes of some laboratories (for discussion see [26]). It should be noted that there were

7 U. Ludewig et al. / FEBS Letters 581 (2007) striking similarities of the function of Rh glycoproteins and TIP2 aquaporins in yeast: heterologous expression of both proteins weakly supported growth on low ammonium in the triple-mepd strain [27,48], but alleviated the toxicity of high MeA [27]. Aquaporin homologs may also facilitate ammonia fluxes at the periarbuscular membrane of mycorrhized plants [50]. Acknowledgements: We thank the Deutsche Forschungsgemeinschaft for financial support (Lu673/7-1) and F. decourcy for critically reading the manuscript. References [1] Miller, A.J. and Cramer, M.D. (2004) Root nitrogen acquisition and assimilation. Plant Soil 274, [2] Loque, D. and von Wiren, N. (2004) Regulatory levels for the transport of ammonium in plant roots. J. Exp. Bot. 55, [3] Soupene, E., He, L., Yan, D. and Kustu, S. (1998) Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein. Proc. Natl. Acad. Sci. 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